Journal: PLoS ONE

Article Title: Exome Sequencing Identifies a Missense Variant in EFEMP1 Co-Segregating in a Family with Autosomal Dominant Primary Open-Angle Glaucoma

doi: 10.1371/journal.pone.0132529

Figure Lengend Snippet: Pedigree and haplotype analysis showing segregation of microsatellite (D2S) markers and single nucleotide variants in EFEMP1 (c.418C>T, c.1033C>T) listed in descending physical order from the short-arm telomere of chromosome 2 (2p-tel). Squares denote males and circles denote females. In the first and second generations, filled symbols denote individuals with confirmed affected status and filled bars denote the disease haplotype. In the third generation, question marks denote individuals of unconfirmed disease status and shaded bars indicate those with the disease haplotype. The trio of individuals I:1, II:4, and II:9 was selected for whole exome sequencing.

Article Snippet: Human EFEMP1 reference and mutant (c.418C>T) cDNA sequences (GenBank accession no. NM_001039348.2) were custom synthesized and directionally sub-cloned into the pReceiver-M13 vector carrying a C-terminal fusion FLAG-tag (GeneCopoeia, Rockville, MD) and the resulting plasmids verified by Sanger sequencing.

Techniques: Sequencing

Journal: PLoS ONE

Article Title: Exome Sequencing Identifies a Missense Variant in EFEMP1 Co-Segregating in a Family with Autosomal Dominant Primary Open-Angle Glaucoma

doi: 10.1371/journal.pone.0132529

Figure Lengend Snippet: ( A ) Schematic of exon organization and protein domains. The gene comprises two non-coding exons (1 & 2) and ten coding exons (3–12) generating at least two transcript variants (2 & 3). Exon 1 and exon 2 are present in transcript variant 2. Exon 2 is skipped in transcript variant 3. Both transcript variants 2 and 3 encode the same 493-amino-acid-protein with 6 calcium-binding (cb) EGF-like domains. The first N-terminal cbEGF-like domain is modified compared with the other five cbEGF-like domains by the insertion of an 88 amino-acid linker region (B). The exon locations of the p.Arg140Trp (p.R140W) and p.Arg345Trp (p.R345W) variants are indicated. ( B ) Amino-acid alignment of the N-terminal cbEGF-like 1 domain (single-letter code) showing cross-species conservation of arginine 140 (R140) located within the 88 amino-acid linker region between conserved cysteine (C) residues (yellow highlight). ( C ) Immunoblot analysis of transfected HEK293T cell-lysates showing expression of wild-type (Arg140) versus mutant (Trp140) EFEMP1-FLAG fusion products. Blots were stripped and re-probed with β-actin to control for sample loading. ( D ) Relative levels of wild-type and mutant EFEMP1-FLAG in transfected cell-lysates normalized to those of β-actin.

Article Snippet: Human EFEMP1 reference and mutant (c.418C>T) cDNA sequences (GenBank accession no. NM_001039348.2) were custom synthesized and directionally sub-cloned into the pReceiver-M13 vector carrying a C-terminal fusion FLAG-tag (GeneCopoeia, Rockville, MD) and the resulting plasmids verified by Sanger sequencing.

Techniques: Variant Assay, Binding Assay, Modification, Western Blot, Transfection, Expressing, Mutagenesis, Control

Journal: bioRxiv

Article Title: Chromosomal instability promotes cell migration and invasion via EFEMP1 secretion into extracellular vesicles

doi: 10.1101/2024.10.21.619397

Figure Lengend Snippet: ( A ) Representative images of EdU incorporation in MDA-MB-231 cells treated with EVs from DMSO or REV treated cells and quantification for three biological replicates. Statistical analysis was performed using two-sided T-tests (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).) ( B ) Cellular component analysis of differentially expressed proteins using Funrich 3.1.4. software. ( C, D ) Migration and invasion of BT549 ( C ) and MDA-MB-231 ( D ) cells treated with DMSO (CIN LOW ) or REV (CIN HIGH ) using trans-well assays. Statistical significance was determined using two-sided T-tests (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Experiments were performed as biological triplicates. Scale bar: 100 µm. ( E ) Gene Ontology (GO) analysis on the proteins enriched in CIN+ EVs compared to CIN-EVs. ( F ) EFEMP1 levels in BT549 cell lysates and BT549 EVs treated with DMSO (CIN LOW ) or REV (CIN HIGH ) detected by Western blot.

Article Snippet: The primary antibodies for Western blots used in this study were EFEMP1 (1:300, Novus Biologicals, NBP1-77040), β-Actin (1:2000, Cell Signaling Technology, 4970 or 3700S), STAT1 (1:1000, Cell Signaling Technology, 9172S), CD63 (1:1000, Novus Biologicals, NBP2-42225), CD81 (1:1000, Novus Biologicals, NB100-65805SS), and Calnexin (1:1000, Novus Biologicals, NB100-1965SS).

Techniques: Software, Migration, Western Blot

Journal: bioRxiv

Article Title: Chromosomal instability promotes cell migration and invasion via EFEMP1 secretion into extracellular vesicles

doi: 10.1101/2024.10.21.619397

Figure Lengend Snippet: ( A ) qPCR quantification of EFEMP1 knockdown efficacy in BT549 cells: Statistical tests were done using a two-sided T-test (n=3; ***, p < 0.001). ( B ) qPCR quantification of EFEMP1 overexpression in BT549 cells. Statistical significance was assessed by a two-sided T-test (n=3; ****, p < 0.0001). ( C ) qPCR quantification of EFEMP1 knockdown efficiency in MDA-MB-231 cells. Statistical analysis using a two-sided T-test (n=3; ***, p < 0.001). ( D ) qPCR quantification of EFEMP1 overexpression. Statistical significance assessed by a two-sided T-test (n=3; ****, p < 0.0001). ( E-F ) Representative images ( E ) and quantification ( F ) of cell migration and invasion of BT549 cells with EFEMP1 overexpression assessed by trans-well assays. Statistical significance was determined by a two-sided T-test (n= 3 biological replicates), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar: 100 µm. ( G, H ) Representative images ( G ) and quantification ( H ) of cell migration and invasion of MDA-MB-231 cells with EFEMP1 overexpression assessed by trans-well assays. Statistical significance was determined by a two-sided T-test (n= 3 biological replicates), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar: 100 µm. ( I ) Representative images (left panel) and quantification (right panel) of scratch assays to quantify migration of BT549 cells with EFEMP1 knockdown. Statistical significance was determined by a two-sided T-test (n= 3 biological replicates), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ( J ) Representative images (left panel) and quantification (right panel) of scratch assays to quantify migration of BT549 cells with EFEMP1 overexpression. Statistical significance was determined by a two-sided T-test (n= 3 biological replicates), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ( K-L ) Representative images ( K ) and quantification ( L ) of cell migration and invasion of BT549 cells with EFEMP1 knockdown assessed by trans-well assays. Statistical significance was determined by a two-sided T-test (n= 3 biological replicates), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar: 100 µm. ( M, N ) Representative images ( M ) and quantification ( N ) of cell migration and invasion of MDA-MB-231 cells with EFEMP1 knockdown (two shRNAs) assessed by trans-well assays. Statistical significance was determined by a two-sided T-test (n= 3 biological replicates), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The primary antibodies for Western blots used in this study were EFEMP1 (1:300, Novus Biologicals, NBP1-77040), β-Actin (1:2000, Cell Signaling Technology, 4970 or 3700S), STAT1 (1:1000, Cell Signaling Technology, 9172S), CD63 (1:1000, Novus Biologicals, NBP2-42225), CD81 (1:1000, Novus Biologicals, NB100-65805SS), and Calnexin (1:1000, Novus Biologicals, NB100-1965SS).

Techniques: Knockdown, Over Expression, Migration

Journal: bioRxiv

Article Title: Chromosomal instability promotes cell migration and invasion via EFEMP1 secretion into extracellular vesicles

doi: 10.1101/2024.10.21.619397

Figure Lengend Snippet: ( A ) Representative images (left panel) and quantification (right panel) of cell migration and invasion of MDA-MB-231 cells treated with EFEMP1 KD EVs assessed by trans-well assays. Statistical significance was determined by a two-sided T-test (n= 3 biological replicates), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar: 100 µm. ( B ) Representative images (left panel) and quantification (right panel) of cell migration and invasion of MDA-MB-231 cells treated with EFEMP1 OEX EVs assessed by transwell assays. Statistical significance was determined by a two-sided T-test (n= 3 biological replicates), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar: 100 µm. ( C ) CD63, CD81, EFEMP1, Calnexin and beta-Actin protein levels in whole cell lysates and EVs from BT549 scramble and EFEMP1 KD cells determined by Western blot. ( D ) CD63, CD81, EFEMP1, Calnexin and beta-Actin protein levels in whole cell lysates and EVs from BT549 control and EFEMP1 OEX cells determined by Western blot. ( E ) CD63, CD81, EFEMP1, Calnexin and beta-Actin protein levels in whole cell lysates and EVs from DMSO- or REV-treated BT549 scramble and EFEMP1 KD cells determined by Western blot. ( F ) CD63, CD81, EFEMP1, Calnexin and beta-Actin protein levels in whole cell lysates and EVs from MDA-MB-231 scramble and EFEMP1 KD cells determined by Western blot. ( G ) CD63, CD81, EFEMP1, Calnexin and beta-Actin protein levels in whole cell lysates and EVs from MDA-MB-231 control and EFEMP1 OEX cells determined by Western blot.

Article Snippet: The primary antibodies for Western blots used in this study were EFEMP1 (1:300, Novus Biologicals, NBP1-77040), β-Actin (1:2000, Cell Signaling Technology, 4970 or 3700S), STAT1 (1:1000, Cell Signaling Technology, 9172S), CD63 (1:1000, Novus Biologicals, NBP2-42225), CD81 (1:1000, Novus Biologicals, NB100-65805SS), and Calnexin (1:1000, Novus Biologicals, NB100-1965SS).

Techniques: Migration, Western Blot, Control

Journal: bioRxiv

Article Title: Chromosomal instability promotes cell migration and invasion via EFEMP1 secretion into extracellular vesicles

doi: 10.1101/2024.10.21.619397

Figure Lengend Snippet: ( A ) Schematic outline of the treatment of BT549 cells with EVs and experimental setup for downstream analyses. ( B, C ) Representative images ( B ) and quantification ( C ) of BT549 migration and invasion Transwell experiments following EFEMP1 knockdown (EFEMP1 KD ) compared to control (scramble) EVs. Statistical significance was determined by a two-sided T-test (n= 3 biological replicates), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar: 100 µm. ( D, E ) Representative images ( D ) and quantification ( E ) of BT549 migration and invasion Transwell experiments following EFEMP1 overexpression (EFEMP1 OEX ) compared to control EVs. Statistical significance was determined by a two-sided T-test (n= 3 biological replicates), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar: 100 µm. (F, G) Representative images ( F ) and quantification ( G ) of migration and invasion of control and CIN HIGH (REV-treated) BT549 cells using Transwell assays under Scramble control and EFEMP1 KD conditions. Statistical significance was determined by a two-sided T-test (n= 3 biological replicates), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar: 100 µm.

Article Snippet: The primary antibodies for Western blots used in this study were EFEMP1 (1:300, Novus Biologicals, NBP1-77040), β-Actin (1:2000, Cell Signaling Technology, 4970 or 3700S), STAT1 (1:1000, Cell Signaling Technology, 9172S), CD63 (1:1000, Novus Biologicals, NBP2-42225), CD81 (1:1000, Novus Biologicals, NB100-65805SS), and Calnexin (1:1000, Novus Biologicals, NB100-1965SS).

Techniques: Migration, Knockdown, Control, Over Expression

Journal: bioRxiv

Article Title: Chromosomal instability promotes cell migration and invasion via EFEMP1 secretion into extracellular vesicles

doi: 10.1101/2024.10.21.619397

Figure Lengend Snippet: ( A ) Venn diagram of RNA sequencing analysis illustrating the downregulation of 29 genes associated with migration and invasion in BT549 cells treated with EFEMP1 KD EVs. The gene sets for migration (166 genes) and invasion (97 genes) were derived from the Cancer Single-cell State Atlas, underscoring the significant role of EFEMP1 in regulating cellular migration and invasion patterns. ( http://biocc.hrbmu.edu.cn/CancerSEA/goDownload ). ( B ) KEGG pathway analysis of the 290 deregulated genes in EFEMP KD cells. ( C ) Molecular Functions (MF) pathway analysis of the 290 deregulated genes in EFEMP KD cells. ( D) Schematic outline of the cell adhesion assay. ( E ) Quantification of cell adhesion of BT549 cells treated with EVs-derived from EFEMP1 KD cells. ( F ) Quantification of cell adhesion of BT549 cells treated with EVs-derived from EFEMP1 OEX cells.

Article Snippet: The primary antibodies for Western blots used in this study were EFEMP1 (1:300, Novus Biologicals, NBP1-77040), β-Actin (1:2000, Cell Signaling Technology, 4970 or 3700S), STAT1 (1:1000, Cell Signaling Technology, 9172S), CD63 (1:1000, Novus Biologicals, NBP2-42225), CD81 (1:1000, Novus Biologicals, NB100-65805SS), and Calnexin (1:1000, Novus Biologicals, NB100-1965SS).

Techniques: RNA Sequencing Assay, Migration, Derivative Assay, Cell Adhesion Assay

Journal: bioRxiv

Article Title: Chromosomal instability promotes cell migration and invasion via EFEMP1 secretion into extracellular vesicles

doi: 10.1101/2024.10.21.619397

Figure Lengend Snippet: ( A ) Correlation analysis of EFEMP1 and STAT1 mRNA expression using DepMap data. ( B ) Correlation analysis of EFEMP1 and STAT1 mRNA expression in DepMap-included breast cancer cell lines. ( C ) qPCR quantification of IL6, IL8, CXCL1, CXCL10, EFEMP1, STAT1, STAT3, CD63 and CYLD in BT549 WT and STAT1 KO cells. Statistical analysis was done using two-sided T-test (N = 3; *p< 0.05, **, p< 0.01, ***p < 0.001, ****p < 0.0001). ( D, E ) Representative images ( D ) and quantification ( E ) of cell migration and invasion of BT549 cells co-cultured with EVs isolated from WT of STAT1 KO BT549 cells, treated with DMSO or REV, assessed by transwell assays. Statistical significance was determined by a two-sided T-test (n= 3 biological replicates), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar: 100 µm. ( F, G ) Representative images ( F ) and quantification ( G ) of cell migration and invasion of BT549 cells co-cultured with EVs isolated from STAT1 KO BT549 cells with or without EFEMP1 overexpression, assessed by transwell assays. Statistical significance was determined by a two-sided T-test (n= 3 biological replicates), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar: 100 µm.

Article Snippet: The primary antibodies for Western blots used in this study were EFEMP1 (1:300, Novus Biologicals, NBP1-77040), β-Actin (1:2000, Cell Signaling Technology, 4970 or 3700S), STAT1 (1:1000, Cell Signaling Technology, 9172S), CD63 (1:1000, Novus Biologicals, NBP2-42225), CD81 (1:1000, Novus Biologicals, NB100-65805SS), and Calnexin (1:1000, Novus Biologicals, NB100-1965SS).

Techniques: Expressing, Migration, Cell Culture, Isolation, Over Expression

Journal: bioRxiv

Article Title: Chromosomal instability promotes cell migration and invasion via EFEMP1 secretion into extracellular vesicles

doi: 10.1101/2024.10.21.619397

Figure Lengend Snippet: ( A ) CD63, CD81, EFEMP1, Calnexin and beta-Actin protein levels in whole cell lysates and EVs from BT549 and STAT KO BT549 cells treated with DMSO or REV determined by Western blot. ( B ) CD63, CD81, EFEMP1, Calnexin and beta-Actin protein levels in whole cell lysates and EVs from STAT KO BT549 cells treated with or without EFEMP1 overexpression determined by Western blot.

Article Snippet: The primary antibodies for Western blots used in this study were EFEMP1 (1:300, Novus Biologicals, NBP1-77040), β-Actin (1:2000, Cell Signaling Technology, 4970 or 3700S), STAT1 (1:1000, Cell Signaling Technology, 9172S), CD63 (1:1000, Novus Biologicals, NBP2-42225), CD81 (1:1000, Novus Biologicals, NB100-65805SS), and Calnexin (1:1000, Novus Biologicals, NB100-1965SS).

Techniques: Western Blot, Over Expression

Journal: bioRxiv

Article Title: Chromosomal instability promotes cell migration and invasion via EFEMP1 secretion into extracellular vesicles

doi: 10.1101/2024.10.21.619397

Figure Lengend Snippet: ( A ) Experimental setup for investigating the invasive behaviour of EV-treated MDA-MB-231 cells in zebrafish embryos. Xenografted embryos were monitored until 1-day post-transplantation (dpt). ( B ) Representative images of embryos xenografted with MDA-MB-231 cells treated with control (left column), EFEMP1 KD EVs (right column), or EFEMP1 OEX EVs (bottom rows) at 1 dpt. Arrowheads point to migrated MDA-MB-231 cells. Scale bar: 200 µm. ( C ) Quantification of MDA-MB-231 cell migration treated with EVs as indicated in large number of zebrafish embryos. Significance was tested using a chi-square test (****, p=3.4084×10 -30 ). ( D ) Quantification of EV-treated MDA-MB-231 cell migration toward the zebrafish embryo’s tail region. Cells were treated with control or EFEMP1 KD EVs as indicated. Significance between groups (control or EFEMP KD EVs) was tested using a chi-square test and migration patterns (categorized as 0, 1-4, and >4 cells migrated cells). p=2.0401×10 -25 . ( E ) Quantification of EV-treated MDA-MB-231 cell migration toward the zebrafish embryo’s tail region. Cells were treated with control or EFEMP1 OEX EVs as indicated. Significance between groups (control or EFEMP KD EVs) was tested using a chi-square test and migration patterns (categorized as 0, 1-4, and >4 cells migrated cells). p=0.0382.

Article Snippet: The primary antibodies for Western blots used in this study were EFEMP1 (1:300, Novus Biologicals, NBP1-77040), β-Actin (1:2000, Cell Signaling Technology, 4970 or 3700S), STAT1 (1:1000, Cell Signaling Technology, 9172S), CD63 (1:1000, Novus Biologicals, NBP2-42225), CD81 (1:1000, Novus Biologicals, NB100-65805SS), and Calnexin (1:1000, Novus Biologicals, NB100-1965SS).

Techniques: Transplantation Assay, Control, Migration

Journal: bioRxiv

Article Title: Chromosomal instability promotes cell migration and invasion via EFEMP1 secretion into extracellular vesicles

doi: 10.1101/2024.10.21.619397

Figure Lengend Snippet: ( A-B ) Scatter plots showing the correlation between EFEMP1 expression and aneuploidy score in DepMap included cancer cell lines ( A ) or DepMap included breast cancer cell lines ( B ). ( C ) Kaplan-Meier survival curves for TGCA-included breast cancer patients, showing overall survival for grade 3 (blue) ( n = 952), grade 2 (yellow) ( n=771 ) and grade 1 (tangerine) ( n=169 ) breast cancer. ( D-F ) Kaplan-Meier survival curves for TGCA-included breast cancer patients stratified for low or high EFEMP1 expression per grade (grade I, D ; grade II, E , grade III, F ). Significant differences between EFEMP1 expression groups were tested using a Log-rank Test.

Article Snippet: The primary antibodies for Western blots used in this study were EFEMP1 (1:300, Novus Biologicals, NBP1-77040), β-Actin (1:2000, Cell Signaling Technology, 4970 or 3700S), STAT1 (1:1000, Cell Signaling Technology, 9172S), CD63 (1:1000, Novus Biologicals, NBP2-42225), CD81 (1:1000, Novus Biologicals, NB100-65805SS), and Calnexin (1:1000, Novus Biologicals, NB100-1965SS).

Techniques: Expressing

Journal: Molecular Cancer Research

Article Title: EFEMP1 Expression Promotes In vivo Tumor Growth in Human Pancreatic Adenocarcinoma

doi: 10.1158/1541-7786.mcr-08-0132

Figure Lengend Snippet: FIGURE 1. Expression of EFEMP1 in vivo. Immunofluorescence of EFEMP1 expression in vivo in tumors originating from FG (A) and L3.6pl cells (B) 37 d after implantation. Insets, DAPI counterstain. Confocal microscopy of EFEMP1 expression in cultured FG cells (C) and L3.6pl cells (D). EFEMP1 staining (green) is increased in L3.6pl cells and follows a homogeneous perinuclear pattern. Bars, 50 Am.

Article Snippet: © 2009 American Association for Cancermcr.aacrjournals.org Downloaded from Cloning and Expression of the EFEMP1 Gene The coding sequence of EFEMP1 cDNA (OriGene Technologies) was amplified by PCR using proofreading Pfx50 DNA polymerase (Invitrogen) and inserted into the expression vector pcDNA 3.2 GW/V5 (Invitrogen).

Techniques: Expressing, In Vivo, Immunofluorescence, Confocal Microscopy, Cell Culture, Staining

Journal: Molecular Cancer Research

Article Title: EFEMP1 Expression Promotes In vivo Tumor Growth in Human Pancreatic Adenocarcinoma

doi: 10.1158/1541-7786.mcr-08-0132

Figure Lengend Snippet: FIGURE 2. Regulation of EFEMP1 expression. Relative expression of EFEMP1 in L3.6pl cells, determined by quantitative RT-PCR. Treatment with IFN-a results in a marked up-regulation of EFEMP1 expression that is counteracted by the NF-nB inhibitor BAY11-7082. Representative data from three independent experiments are shown.

Article Snippet: © 2009 American Association for Cancermcr.aacrjournals.org Downloaded from Cloning and Expression of the EFEMP1 Gene The coding sequence of EFEMP1 cDNA (OriGene Technologies) was amplified by PCR using proofreading Pfx50 DNA polymerase (Invitrogen) and inserted into the expression vector pcDNA 3.2 GW/V5 (Invitrogen).

Techniques: Expressing, Quantitative RT-PCR

Journal: Molecular Cancer Research

Article Title: EFEMP1 Expression Promotes In vivo Tumor Growth in Human Pancreatic Adenocarcinoma

doi: 10.1158/1541-7786.mcr-08-0132

Figure Lengend Snippet: FIGURE 3. In vivo growth of EFEMP1-overexpressing tumors. A. Expression levels of EFEMP1 protein in FG, FG-EFEMP1, and L3.6pl cells. B. Orthotopic pancreatic tumor growth after injection of FG, FG-EFEMP1, and L3.6pl cells. FG-EFEMP1 versus FG: x, P < 0.05; *, P < 0.01. C. Tumor weight on necropsy 28 d after tumor cell injection. FG versus FG-EFEMP1, and FG-EFEMP1 versus L3.6pl; *, P < 0.01. D. Macroscopic incidence of hepatic and lymphogenic metastases (n = 10). E to G. H&E staining of FG (E), FG-EFEMP1 (F), and L3.6pl (G) tumors grown orthotopically. Necrosis (black arrows) and fibrotic tissue (white arrows) both are most pronounced in L3.6pl tumors. Bars, 200 Am.

Article Snippet: © 2009 American Association for Cancermcr.aacrjournals.org Downloaded from Cloning and Expression of the EFEMP1 Gene The coding sequence of EFEMP1 cDNA (OriGene Technologies) was amplified by PCR using proofreading Pfx50 DNA polymerase (Invitrogen) and inserted into the expression vector pcDNA 3.2 GW/V5 (Invitrogen).

Techniques: In Vivo, Expressing, Injection, Staining

Journal: Molecular Cancer Research

Article Title: EFEMP1 Expression Promotes In vivo Tumor Growth in Human Pancreatic Adenocarcinoma

doi: 10.1158/1541-7786.mcr-08-0132

Figure Lengend Snippet: FIGURE 4. EFEMP1 induced angiogenesis. A. In FG-EFEMP1 transfectants, a significant increase in VEGF production was observed (*, P < 0.01 in FG vector controls versus FG-EFEMP1). Notably, no further stimulation of VEGF production was seen in EFEMP1-transfected L3.6pl cells compared with L3.6pl vector controls. B. Proliferation of HUVECs induced by VEGF-A (50 ng/mL) and EFEMP1 protein (10 and 100 ng/mL). No significant direct effect of EFEMP1 protein on the proliferation was observed. VEGF-A versus medium control; *, P < 0.01. C. Migration of HUVECs induced by VEGF-A (50 ng/mL) and EFEMP1 protein (10 and 100 ng/mL) in a modified Boyden chamber assay. Again, there was no significant direct effect of EFEMP1 protein. VEGF-A versus medium control; *, P < 0.01. D. Microvascular density is increased in tumors grown from FG-EFEMP1 cells compared with FG (*, P < 0.05). E to G. CD31 staining of tumor specimens grown from FG (E), FG-EFEMP1 (F), and L3.6pl cells (G). Bars, 100 Am.

Article Snippet: © 2009 American Association for Cancermcr.aacrjournals.org Downloaded from Cloning and Expression of the EFEMP1 Gene The coding sequence of EFEMP1 cDNA (OriGene Technologies) was amplified by PCR using proofreading Pfx50 DNA polymerase (Invitrogen) and inserted into the expression vector pcDNA 3.2 GW/V5 (Invitrogen).

Techniques: Plasmid Preparation, Transfection, Control, Migration, Modification, Boyden Chamber Assay, Staining

Journal: Molecular Cancer Research

Article Title: EFEMP1 Expression Promotes In vivo Tumor Growth in Human Pancreatic Adenocarcinoma

doi: 10.1158/1541-7786.mcr-08-0132

Figure Lengend Snippet: FIGURE 5. Suppression of apoptosis by EFEMP1 expression in vitro and in vivo. Under serum starvation conditions, a FACS scan of FG vector controls (A) shows 83.4% apoptotic tumor cells, whereas the proportion of apoptotic FG-EFEMP1 cells (B) is reduced (48.0%). Data from a typical experiment are shown. C. Proportions of apoptotic (solid bar), G0-G1–phase (dense bands), S-phase (sparse bands), and mitotic cells (white) of FG, FG-EFEMP1, and L3.6pl cells cultivated under serum starvation conditions. D. TUNEL staining of apoptotic tumor cells in xenografts of FG, FG-EFEMP1, and L3.6pl cells. Bar, 100 Am.

Article Snippet: © 2009 American Association for Cancermcr.aacrjournals.org Downloaded from Cloning and Expression of the EFEMP1 Gene The coding sequence of EFEMP1 cDNA (OriGene Technologies) was amplified by PCR using proofreading Pfx50 DNA polymerase (Invitrogen) and inserted into the expression vector pcDNA 3.2 GW/V5 (Invitrogen).

Techniques: Expressing, In Vitro, In Vivo, Plasmid Preparation, TUNEL Assay, Staining

Journal: Molecular Cancer Research

Article Title: EFEMP1 Expression Promotes In vivo Tumor Growth in Human Pancreatic Adenocarcinoma

doi: 10.1158/1541-7786.mcr-08-0132

Figure Lengend Snippet: FIGURE 6. Relative expres- sion of EFEMP1 mRNA in human pancreatic ductal adenocarcino- ma specimens. EFEMP1 mRNA expression in the tumor and surrounding normal pancreatic tissue was compared, and indi- vidual samples are shown. EFEMP1 mRNA expression was up-regulated in 13 of 15 tumors. Columns, mean relative mRNA expression of quantitative RT- PCR done in triplicate.

Article Snippet: © 2009 American Association for Cancermcr.aacrjournals.org Downloaded from Cloning and Expression of the EFEMP1 Gene The coding sequence of EFEMP1 cDNA (OriGene Technologies) was amplified by PCR using proofreading Pfx50 DNA polymerase (Invitrogen) and inserted into the expression vector pcDNA 3.2 GW/V5 (Invitrogen).

Techniques: Expressing, Quantitative RT-PCR